One of the major physiological roles of insulin is to stimulate glucose transport into insulin-sensitive cells and tissues by inducing the translocation of the major insulin responsive glucose transporter, GLUT4, from an intracellular compartment to the plasma membrane. Secretion of insulin from the beta cells in the pancreas thus tightly regulates glucose homeostasis, which is critical for normal physiologic maintenance in higher animals.
Insulin resistance, which is commonly associated with prevalent type 2 diabetes, is a state in which target cells fail to respond to normal levels of circulating insulin. See, e.g. Saltiel et al., Nature 414: 799-806 (2001). This lack of response, in turn, results in hyperinsulinemia to compensate for the resistance to insulin in the prediabetic state. Subsequently, hyperglycemia develops due to the failure of the pancreatic beta cells to produce and secrete enough insulin to compensate for the imbalance in glucose metabolism. Type 2 diabetes is the most common form of the disease, affecting 16 million people in the United States alone. (Source: American Diabetes Association). Roughly one-third of these people remain undiagnosed. (Source: id.)
At the molecular level, insulin resistance may result from mutations or posttranslational modifications of the insulin receptor or any of its downstream targets. The identification of simple molecular explanations for insulin resistance and type 2 diabetes has so far proven difficult. Understanding the basic mechanisms of insulin resistance at the molecular level could have a great impact on finding a cure for a chronic disease like type-2 diabetes and insulin resistance that develops in other conditions such as chronic obesity and acute trauma.
The insulin receptor (IR) is a transmembrane glycoprotein, comprising an extracellular insulin-binding domain and a transmembrane tyrosine protein kinase domain that undergoes autophosphorylation following insulin binding. Auto-phosphorylation in turn activates the IR intrinsic tyrosine kinase activity and triggers phosphorylation of numerous downstream targets that ultimately mediate insulin's several biological effects.
The major targets of insulin receptor kinase are the insulin-receptor substrate (IRS) proteins IRS-1, IRS-2, IRS-3, IRS-4, the adaptor proteins Shc, Gab1, APS, p60Dok, SIRPS and c-Cbl. See, e.g. White, M. F., Mol. Cell. Biochem. 182: 3-11 (1998); Kahn et al., U.S. Pat. No. 5,621,075, Issued Apr. 15, 1997; Holgado-Madruga, et al., Nature 379: 560-564 (1996); Sasaoka et al., J. Biol. Chem. 269: 13689-694 (1994); Moodie et al., J. Biol. Chem. 274:11186-193 (1999). The C-terminal region of IRS proteins contains multiple tyrosine phosphorylation motifs that serve as docking sites for many SH2 domain-containing proteins, such as the p85 regulatory subunit of PI3K, which mediate many of the down-stream biological actions of insulin. See, e.g. White (1998), supra.
The relative roles of the different IRS proteins in insulin signaling and diabetes have been intensively studied. Gene targeting experiments have revealed that IRS proteins are essential for normal development and metabolism. See, e.g. Saltiel et al., Trends Cell Biol. 12: 65-71 (2002). For example, mice lacking IRS-1 grow poorly in utero and remain small throughout life, but diabetes does not develop because insulin secretion increases to compensate for a mild insulin resistance. In contrast, IRS-2 null mice develop insulin resistance and beta cell failure, and die from type 2-like diabetes. These results not only suggest a critical role of IRS proteins in mediating insulin action, but also indicate that understanding the regulation of IRS proteins can provide important clues as to the causes of insulin resistance.
Protein phosphorylation is an important mechanism by which the activity of the insulin-signaling pathway, as with most signaling pathways, is regulated. A major negative regulatory mechanism for insulin action has been attributed to agents that enhance serine or threonine (Ser/Thr) phosphorylation of either the IR itself, or of its downstream effectors. Ser/Thr phosphorylation reduces the tyrosine kinase activity of the IR, and thus its ability to phosphorylate substrate proteins. For example, insulin's counter regulators, such as epinephrine and glucagons, increase cAMP levels in the cell, thereby activating the cAMP-dependent protein kinase (PKA) and increasing the Ser/Thr phosphorylation of the insulin receptor, which results in an insulin-resistant state. Similarly, the general inhibitor of protein phosphatases, okadaic acid, inhibits tyrosine phosphorylation of IRS-1, while increasing its phosphorylated Ser/Thr content. Tumor necrosis factor-α, a known mediator of insulin resistance during infection, tumor cachexia, and obesity all cause similar effects.
It has been postulated that phosphorylation of serine residues significantly reduces the ability of IRS-1 and IRS-2 to interact with, and become, tyrosine phosphorylated by the IR. See, e.g. White (1998), supra.; Zick Y., Trends Cell Biol. 11: 437-441 (2001); Saltiel et al. (2002), supra. Phosphorylation of IRS at Ser/Thr residues and the consequent inactivation of insulin signaling can be triggered by prolonged exposure to insulin itself or by cross-desensitization with other factors that provoke IRS phosphorylation, e.g. PDGF, IGF-1 endothelin, or TNFα.
However, there is mounting evidence indicating that each stimulus can desensitize IRS-1 and IRS-2 function through a different mechanism, implying the phosphorylation of different sites in the IRS proteins. Indeed, IRS proteins contain over 30 potential Ser/Thr phosphorylation sites for kinases like PKA, PKC, mitogen-activated protein kinase (MAPK), Akt (PKB) and others. See, e.g. White (1998), supra.; Zick, supra.; Saltiel et al. (2002), supra. For example, stimulators of PKC, such as phorbol esters or endothelin, induce the activity of MAPK, which then phosphorylates IRS-1 at Ser612. See, e.g. Jiang et al., Diabetes 48: 1120-1130(1999); Mothe et al., J. Biol. Chem. 271:9351-9356(1996). This phosphorylation event reduces IRS-1 tyrosine phosphorylation by the IR, as well as IRS-1 association with PI3K. In contrast, the inhibitory effects of PDGF were reported to not require Ser612, but instead phosphorylation of three other serine residues (632, 662, and 731) were involved through a mechanism implicating PI3K/Akt and mTOR pathway. See Li J., J. Biol. Chem. 274: 9351-9356 (1999). Phospho-specific antibodies to certain of these IRS-1 phosphorylation sites are commercially available. (See, e.g. Upstate Biotechnology, Inc., Cat. No. 07-247(Ser307); Cell Signaling Technology, Inc., Cat. Nos. 2388, 2386 (Ser 636/639 and 612); BioSource, Inc., Cat. No. 44-550 (Ser616)).
The protein kinase Akt, however, has been implicated in positively modulating IRS function by preventing its rapid tyrosine phosphorylation. See Zick, supra. The action of Akt appears to involve four possible serine residues in human IRS (270, 307, 330, and 383), but the evidence on which of these sites is essential for negative or positive regulation by Akt is inconclusive. Other recent studies suggest that Ser312 (307 in the mouse) in IRS-1 is actually regulated by TNF-α through activation and direct phosphorylation by the Jun-terminal kinase (JNK), a kinase of the MAPK family. See, e.g. Aguirre et al., J. Biol. Chem. 275: 9047-9054 (2000); Rui et al., J. Clin. Invest. 107. 181-189 (2001); Aguirre et al., J. Biol. Chem. 277:1531-1537 (2002). This finding could explain the well-documented insulin resistance that is provoked by acute stress and mediated through TNF-α action.
Taken together, these results underscore the important role that IRS-1 Ser/Thr phosphorylation plays in type 2 diabetes. However, the precise mechanisms by which particular signaling events are mediated by IRS-1 phosphorylation remain unclear, and the serine or threonine residues relevant to such mechanisms remain unidentified. For example, chronic and acute elevation of plasma free fatty acid is commonly linked to impaired insulin-mediated glucose uptake. See Griffin et al., Diabetes 48:1270-1274 (1999). The mechanisms underlying these changes in glucose transport are unknown, but may include changes in insulin signaling. It has been shown that protein kinase C (PKC) theta protein levels, one of the major PKC isoforms expressed in skeletal muscle, are elevated in insulin resistant humans and rats. Moreover, PKC theta activity is enhanced by elevated plasma free-fatty acids. See Itani et al., Metabolism 50: 553-557 (2001); Qu et al., J. Endocrinol. 162: 207-214 (1999); Chalfant et al., Endocrinology 141: 2773-2778 (2000). PKC theta thus represents a potential therapeutic target for modulating insulin signaling in obesity-driven insulin resistance, but the mechanism of its action remains unclear.
Accordingly, there remains a need for the identification of Ser/Thr phosphorylation sites in IRS-1 and IRS-2 that are essential for the inhibition of insulin signaling leading to insulin resistance and type 2 diabetes. The production of phospho-specific antibodies directed at such sites would greatly facilitate the elucidation of critical phosphorylated Ser/Thr residues in IRS-1 and IRS-2, particularly in the context of the diverse pathological circumstances and pathways that cause insulin resistance. Such antibodies would be valuable tools for the early diagnosis of type-2 diabetes and other conditions involving insulin resistance, as well as for drug discovery programs aimed at identifying new compounds for the restoration of insulin-sensitivity in diabetic individuals.